Experiments in this proposal will attempt to place into biologic context the contributions of p300 and hHR23 proteins to p53 homeostatic regulation by the ubiquitin/proteasome pathway. The p300 coactivator encodes both histone acetylase and ubiquitin ligase functions, and thus acts as a nexus for p53 homeostasis, encoding both positive and negative regulatory functions, p300 interacts with the p53 antagonist and ubiquitin ligase, MDM2, and the two proteins cooperate in the polyubiquitination and rapid proteasome turnover of p53. Under conditions of stress or DNA damage, p300 activates p53 functions through its intrinsic acetylase activity. hHR23 proteasome adaptor proteins also profoundly influence the metabolism and activity of p53 in human cells, hHR23-family proteins protect p53 from degradation, and may integrate ubiquitination and degradation of p53, by forming a complex with MDM2. Moreover, a novel, potentially regulatory or dominant negative third hHR23 family member has been identified which, unlike the known hHR23 family members, is not universally expressed in all cell lines, tumors and tissues. The proposed experimental aims address the specific mechanisms by which p3OO/MDM2 interaction, p300 ubiquitin ligase activity, and hHR23 proteins act as key components in the dynamic physiologic regulation of p53 response to oncogenic stressors, such as ionizing radiation. The role of p300/MDM2 interaction in determining how p300 activity as an activator or antagonist of p53 is balanced, will be examined by mapping of the interaction, and analysis of p53 regulation in cells expressing non-interacting mutant alleles. The specific role of p300 ubiquitin ligase activity in p53 homeostasis will be studied by analysis of loss of function alleles in primary human cells and a knock-in mouse model. The function of the hHR23A and hHR23B proteasome adaptor proteins, and the role of hHR23/MDM2 interaction in p53 regulation, will be analyzed by structure/function analysis of hHR23 proteins in cultured ceils and in a reconstituted in vitro p53 degradation system. A novel, potentially regulatory, hHR23 family member will also be further characterized for its role in DNA damage regulation of p53 stability.